Bottom Line:
Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

f7: Attenuated in vitro and in vivo suppressive activity of Nr4a2-deficient Tregs.(a) FACS analysis of the indicated protein levels in YFP+Foxp3+ and YFP–Foxp3+ Tregs of Nr4a2+/+Foxp3Yfp-Cre/+ or Nr4a2fl/flFoxp3Yfp-Cre/+ mice. For Foxp3, CD25, CTLA-4 and GITR expression levels in non-Treg cells are represented by black lines, as a negative value. Data are representative of three independent experiments (means of duplicate are shown for the positive fractions of Annexin V and Ki67 staining). (b) Comparison of mRNA levels between Tregs in Nr4a2+/+Foxp3Yfp-Cre and Nr4a2fl/flFoxp3Yfp-Cre mice. Expression levels of the indicated mRNA, are presented relative to Hprt expression. Open bars: Nr4a2+/+Foxp3Yfp-Cre Tregs; closed bars: Nr4a2fl/flFoxp3Yfp-Cre Tregs. (c) Top: in vitro suppression assay with CFSE-labeled wild-type CD4+CD25– cells as responders and CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre (open squares) or Nr4a2fl/flFoxp3Yfp-Cre (closed triangles) mice as suppressors. Ratios of responder cells with more than three divisions are presented. Bottom: results in the top panel at 1:1 Treg/Tresp ratio. (d) Body weight of Rag2– mice injected with 5×105 naïve Ly5.1+CD4+ T cells alone or in combination with 3×105 CD4+CD25+YFP+Ly5.2+ Tregs from Nr4a2+/+Foxp3Yfp-Cre or Nr4a2fl/flFoxp3Yfp-Cre mice. Data are pooled from two independent experiments, with seven mice from each sample set total. The ranges of body weights (relative body weights to day 0) of each group at day 21 are Tnaive only: 0.69–0.76; Tnaive+Nr4a2+/+Foxp3Yfp-Cre Tregs: 0.96–1.08; Tnaive+Nr4a2fl/flFoxp3Yfp-Cre Tregs: 0.76–0.88. (e) Expression of Foxp3 and IFN-γ in CD4+ T cells from the spleen and lymph nodes (left panels), and from the colon and caecum lamina propria (right panels) in Rag2– recipients presented in d at day 21. (f) Frequencies of IFN-γ-expressing cells in Foxp3–CD4+ T cells and Foxp3+CD4+ T cells from the spleen and lymph nodes of Rag2– recipients presented in d at day 21. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. Data in b, c are representative of three independent experiments (mean and s.d. of triplicate). *P<0.05 (two-tailed Student's t-test).

Mentions:
To further investigate the roles of Nr4a2 in Tregs we measured expression levels of Treg markers, activation status, cell death and proliferation in YFP+Foxp3+ and YFP–Foxp3+ Tregs in Nr4a2+/+Foxp3YFP-Cre/+ and Nr4a2fl/flFoxp3YFP-Cre/+ mice, which allowed us to compare Nr4a2-deficient and Nr4a2-suficient Tregs in the same mice. We observed decreased levels of Foxp3 and CD25 in YFP+ fraction in Nr4a2fl/flFoxp3YFP-Cre/+ mice (Fig. 7a). Other Treg markers such as CTLA-4 and GITR were expressed at a similar level between Nr4a2-deficient and Nr4a2-sufficient Tregs (Fig. 7a). A similar expression pattern of CD62L indicated equivalent activation status. A similar percentage of cells staining positive for Annexin V and Ki67 indicated an equivalent rate of cell proliferation and death between the Nr4a2-deficient and Nr4a2-sufficient Tregs, further supporting the notion that the insufficiency of the Nr4a2-deficient Tregs observed in Figure 6a,b was not caused by a difference in cell death or proliferation. We also compared the expression levels of Treg-relevant mRNA between Tregs from Nr4a2+/+Foxp3YFP-Cre and Nr4a2fl/flFoxp3YFP-Cre mice. We found decreased expression of Foxp3, indicating that the accelerated loss of Foxp3 in Nr4a2-deficient Tregs was at the mRNA level (Fig. 7b). For other genes, we observed a significant reduction of CD25, TGF-β1 and CCR9.

f7: Attenuated in vitro and in vivo suppressive activity of Nr4a2-deficient Tregs.(a) FACS analysis of the indicated protein levels in YFP+Foxp3+ and YFP–Foxp3+ Tregs of Nr4a2+/+Foxp3Yfp-Cre/+ or Nr4a2fl/flFoxp3Yfp-Cre/+ mice. For Foxp3, CD25, CTLA-4 and GITR expression levels in non-Treg cells are represented by black lines, as a negative value. Data are representative of three independent experiments (means of duplicate are shown for the positive fractions of Annexin V and Ki67 staining). (b) Comparison of mRNA levels between Tregs in Nr4a2+/+Foxp3Yfp-Cre and Nr4a2fl/flFoxp3Yfp-Cre mice. Expression levels of the indicated mRNA, are presented relative to Hprt expression. Open bars: Nr4a2+/+Foxp3Yfp-Cre Tregs; closed bars: Nr4a2fl/flFoxp3Yfp-Cre Tregs. (c) Top: in vitro suppression assay with CFSE-labeled wild-type CD4+CD25– cells as responders and CD4+CD25+YFP+ Tregs from Nr4a2+/+Foxp3Yfp-Cre (open squares) or Nr4a2fl/flFoxp3Yfp-Cre (closed triangles) mice as suppressors. Ratios of responder cells with more than three divisions are presented. Bottom: results in the top panel at 1:1 Treg/Tresp ratio. (d) Body weight of Rag2– mice injected with 5×105 naïve Ly5.1+CD4+ T cells alone or in combination with 3×105 CD4+CD25+YFP+Ly5.2+ Tregs from Nr4a2+/+Foxp3Yfp-Cre or Nr4a2fl/flFoxp3Yfp-Cre mice. Data are pooled from two independent experiments, with seven mice from each sample set total. The ranges of body weights (relative body weights to day 0) of each group at day 21 are Tnaive only: 0.69–0.76; Tnaive+Nr4a2+/+Foxp3Yfp-Cre Tregs: 0.96–1.08; Tnaive+Nr4a2fl/flFoxp3Yfp-Cre Tregs: 0.76–0.88. (e) Expression of Foxp3 and IFN-γ in CD4+ T cells from the spleen and lymph nodes (left panels), and from the colon and caecum lamina propria (right panels) in Rag2– recipients presented in d at day 21. (f) Frequencies of IFN-γ-expressing cells in Foxp3–CD4+ T cells and Foxp3+CD4+ T cells from the spleen and lymph nodes of Rag2– recipients presented in d at day 21. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. Data in b, c are representative of three independent experiments (mean and s.d. of triplicate). *P<0.05 (two-tailed Student's t-test).

Mentions:
To further investigate the roles of Nr4a2 in Tregs we measured expression levels of Treg markers, activation status, cell death and proliferation in YFP+Foxp3+ and YFP–Foxp3+ Tregs in Nr4a2+/+Foxp3YFP-Cre/+ and Nr4a2fl/flFoxp3YFP-Cre/+ mice, which allowed us to compare Nr4a2-deficient and Nr4a2-suficient Tregs in the same mice. We observed decreased levels of Foxp3 and CD25 in YFP+ fraction in Nr4a2fl/flFoxp3YFP-Cre/+ mice (Fig. 7a). Other Treg markers such as CTLA-4 and GITR were expressed at a similar level between Nr4a2-deficient and Nr4a2-sufficient Tregs (Fig. 7a). A similar expression pattern of CD62L indicated equivalent activation status. A similar percentage of cells staining positive for Annexin V and Ki67 indicated an equivalent rate of cell proliferation and death between the Nr4a2-deficient and Nr4a2-sufficient Tregs, further supporting the notion that the insufficiency of the Nr4a2-deficient Tregs observed in Figure 6a,b was not caused by a difference in cell death or proliferation. We also compared the expression levels of Treg-relevant mRNA between Tregs from Nr4a2+/+Foxp3YFP-Cre and Nr4a2fl/flFoxp3YFP-Cre mice. We found decreased expression of Foxp3, indicating that the accelerated loss of Foxp3 in Nr4a2-deficient Tregs was at the mRNA level (Fig. 7b). For other genes, we observed a significant reduction of CD25, TGF-β1 and CCR9.

Bottom Line:
Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.